The intestinal epithelium plays a crucial role in barrier function. These cells are often injured and progresses to death in inflammatory bowel disease (IBD)-like disorders. Epithelial cell loss typically occurs via a mixture of cell death phenotypes such as apoptosis or necroptosis, but is dominated by lysosomal-mediated (LM) necrosis when the insult is severe. Clinically, LM necrosis is manifest by large segments of the epithelial lining sloughing into the intestinal lumen. Preservation of normal epithelial cell viability and function will help ameliorate the complications of IBD-like disorders. Initially perceived as haphazard and irreversible, we show that LM necrosis follows a molecular program and is reversible. We hypothesize that the development of a high-throughput, high-content in vivo screen (HTS/HCS) for small molecule antidotes to LM necrosis will prime the preclinical drug discovery pipeline for a form of intestinal epithelial cell death that currently lacks any means of chemoprophylaxis. Unfortunately, the expense and complexity of mammalian systems make them unsuitable for HTS/HCS formats. Moreover, these systems present with mixed cell death phenotypes, thereby complicating the isolation of pure LM necrosis inhibitors. We identified a C. elegans strain null for an inhibitor of lysosomal cysteine proteases. These animals are highly and exclusively susceptible to LM intestinal necrosis when stressed by many of the noxious stimuli occurring in IBD-like disorders. This cell death pathway is evolutionarily conserved, as fetal intestinal cell explant cultures from mice null for the homologous cysteine protease inhibitor also preferentially undergo LM intestinal necrosis, even when exposed to apoptotic or necroptotic stimuli. Our laboratory pioneered the use of mutant C. elegans strains to conduct live-animal, HTS/HCS drug discovery campaigns (US Patent US8809617 B2). Compounds from these screens have already proved effective in analogous mammalian models of disease. Using this robust technology, we propose to use our mutant C. elegans strain to perfect an HTS/HCS assay for LM necrosis and conduct a large-scale screen for small molecule inhibitors of intestinal epithelial LM necrosis. Promising hits have already been identified in a small pilot screen using a library of FDA-approved drugs. Hits will be validated using a number of secondary assays involving nematodes, mammalian cell lines and a mouse intestinal epithelial injury protocol that models human necrotizing enterocolitis. The ability to conduct a live-animal, LM necrosis phenotype-based screens for new therapeutics is unprecedented for episodes of IBD-like diseases marked by extensive epithelial cell injury. This proposal was prepared in response to PA-16-374, Assay Development and Screening to Discover Therapeutic or Imaging Agents for Diseases of interest to NIDDK.